4-(isoquinolin-1-yl) piperazine-1-carboxylic acid esters

ABSTRACT

4-(ISOQUINOLIN-1-YL)-PIPERAZINE-1-CARBOXYLIC ACID, ESTERS AND 1-AMINO-6,7-DIALKOXYISOQUINOLINES AND THEIR PHARMACEUTICALLY-ACCEPTABLE ACID ADDITION SALTS. COMPOUNDS MANIFEST BRONCHODILATOR ACTIVITY AND ANTIHYPERTENSIVE RESPONSE WITH MINIMAL ADVERSE EFFECTS UPON ADMINISTRATION TO AFFLICTED SUBJECTS.

3,702,849 4-(ISOQUINOLlN-1-YL) PIPERAZINE-l- CARBOXYLIC ACID ESTERSTimothy H. Cronin, Niantic, and Hans-Jurgen E. Hess,

Old Lyme, Cnn., assignors to Pfizer Inc., New York,

No Drawing. Application Oct. 26, 1967, Ser. No. 678,191, now Patent No.3,517,005, dated Jan. 23, 1970, which is a continuation-in-part ofapplication Ser. N 0. 590,494, Oct. 31, 1966. Divided and thisapplication Mar. 17, 1970, Ser. No. 20,381

Int. Cl. C07d 51/70 U.S. Cl. 260-268 C 4 Claims CROSS REFERENCE TORELATED APPLICATIONS This application is a division of application Ser.No. 678,191, filed Oct. 26, 1967 (now U.S. Pat. No. 3,517,- 005), which,in turn, is a continuation-in-part of application Ser. No. 590,494,filed Oct. 31, 1966 and now abandoned.

BACKGROUND OF THE INVENTION This invention relates to certain novelorganic nitrogen compounds, and more particularly, it is concerned withvarious new and useful organic heterocyclic nitrogen compounds which arevaluable in alleviating bronchoconstriction in afllicted subjects and inreducing the blood pressure of hypertensive subjects.

Treatment of subjects sufiering from bronchoconstriction or hypertensionrequires that the therapeutic agent effectively cause gronchodilation orlower the blood pressure of the treated subject at dosage levels whichdo not cause other undesirbale effects in the subject. The compounds ofthis invention manifest bronchodilatory and/or hypotensive activity atdosage levels at which no adverse efiects are manifested in the treatedindividual.

SUMMARY OF THE INVENTION Among the compounds of this invention are'thoseof 14 R1: VI and their pharmaceutically-acceptable acid addition salts.

"United States Patent 0 ice In these compounds of Formula VI, A and Bmay each be alkoxy containing from 1 to 5 carbon atoms, hydrogen,hydroxy, or methyl. When A or B are hydrogen, the other is neverhydrogen. A and B, taken together, may be benzo or alkylenedioxy havingup to 4 carbon atoms. R, may be H or alkyl containing from 1 to 6 carbonatoms. R R R and R each may be H, alkyl containing from 1 to 4 carbonatoms or hydroxymethyl and R and R taken together, may be cycloalkylhaving from 4 to 7 carbon atoms. I may be H, hydroxy, formyloxy, acyloxycontaining from 2 to 5 carbon atoms, aroyloxy containing up to 11 carbonatoms, alkoxy containing from 1 to 4 carbon atoms, aryloxy containing upto 10 carbon atoms, chloro, bromo, formamido, alkylamido having up to 4carbon atoms in the alkyl group, .arylamido having up to 10 carbon atomsin the aryl group or amino, containing up to two substituents. Eachamino substituent may be hydrogen, alkyl having up to 4 carbon atoms oraryl containing up to 10 carbon atoms. The two substituents, takentogether, may be cycloalkyl containing from 4 to 7 carbon atoms.

These compounds are useful as bronchodilators and as smooth musclerelaxants. Of particular interest as bronchodilators are the compound4-(6,7-dimethoxyquin- 01in 4 yl) piperazine 1 carboxylic acid, isobutylester, and the compound 4-(6,7-dimethoxyquinolin-4-yl)- piperazine 1carboxylic acid, 2 methyl 2 hydroxypropyl ester.

Other compounds of this invention are those of the formulae:

R1 VII and and their pharmaceutically-acceptable acid addition salts.

In the compounds of Formulae VII, VIII and IX, A and B may each bealkoxy containing from 1 to carbon atoms, hydrogen, hydroxy, or methyl.When A or B are hydrogen, the other is never hydrogen. A and B, takentogether may be benzo or alkylenedioxy having up to 4 carbon atoms. R isH or alkyl containing from 1 to 6 carbon atoms. R and R may each be H,alkyl or alkenyl containing up to 6 carbon atoms, aryl hydrocarboncontaining up to carbon atoms, aralkyl hydrocarbon containing up to 3carbon atoms in the alkyl moiety and 7 carbon atoms in the aryl moiety,or p-hydroxyethyl. When R and R are aryl or aralkyl, the aryl moiety maybe substituted with up to 3 substituents which may be halogen or alkoxycontaining up to 4 carbon atoms. R and R taken together, may becycloalkyl containing from 3 to 7 carbon atoms.

G may be oxygen, sulfur, amino, alkylamino having up to 6 carbon atomsin the alkyl group, alkenylamino having from 3 to 6 carbon atoms in thealkenyl moiety, unsubstituted aryl hydrocarbon amino having up to 10carbon atoms in the aryl moiety, acylamino having up to 6 carbon atomsin the acyl moiety, aroylamino having up to 11 carbon atoms in the aroylmoiety, carbalkoxyamino having up to 6 carbon atoms in the alkyl moiety,carbalkenyloxyamino having up to 6 carbon atoms in the alkenyl moiety orcarbaryloxyamino having up to 10 carbon atoms in the aryl moiety. R R Rand R may each be H, alkyl containing from 1 to 4 carbon atoms orhydroxymethyl. R and R taken together, may be cycloalkyl having from 4to 7 carbon atoms.

I may be H, hydroxy, formyloxy, acyloxy containing from 2 to 5 carbonatoms, aroyloxy containing up to 11 carbon atoms, alkoxy containing from1 to 4 carbon atoms, aryloxy containing up to 10 carbon atoms, chloro,bromo, formarnido, alkylamino having up to 4 carbon atoms in the alkylgroup, arylamido having up to 10 carbon atoms in the aryl group oramino, containing up to two substituents. Each amino substituent may behydrogen, alkyl having up to 4 carbon atoms or aryl containing up to 10carbon atoms. The two substituents, taken together, may be cycloalkylcontaining from 4 to 7 carbon atoms.

These compounds of Formulae VII, VIII and IX are useful asbronchodilators and as smooth muscle relaxants. Particularly preferredas bronchodilators are the compound 4-(6,7 dimethoxyisoquinolin 1yl)-piperazinel-carboxylic acid isobutyl ester, the compound4-(6,7-dimethoxyisoquinolin 1 yl) piperazine l carboxylic acid, ethylester, the compound 4 (6,7 dimethoxyisoquinolin 1 yl)-piperazine 1carboxylic acid, Z-methyl- 2 hydroxypropyl ester, and the compound 4ethylamino 6,7 dimethoxyisoquinoline.

DETAILED DESCRIPTION OF THE INVENTION The compounds of Formula VI areprepared from the corresponding 4-ch1oro-6,7-substituted quinolines.These latter compounds are prepared by the method given by B. Riegel etal., J. Am. Chem. Soc., 68, 1264 (1946). The nature of the 6- and7-substituents is determined by the original 3,4 (ii-substituted anilinewhich is used to prepare the 6,7-substituted quinoline. The nature ofthe Z-substituent is determined by the malonic ester which is used tocyclize the 3,4-disubstituted aniline.

The compounds of Formula VI are prepared by reacting the 4 chloro 6,7substituted quinoline with an appropriate piperazine-l-carboxylic acidester. This reaction may be carried out in an appropriate aqueous ororganic solvent and, while ethanol is a preferred solvent, other polarsolvents such as dimethylformamide, dioxane, tetrahydrofuran or methanolmay be used. A molar excess of the piperazine acid ester may beemployed. The reaction mixture is heated at a temperature from 100 C. to170. for a period ranging from one to sixteen hours. Preferred reactiontimes are from 1.5 to 2.5 hours and preferred temperatures range from C.to C., where ethanol is the solvent.

An alternative procedure may be used which involves the preparation of 4piperazinyl quinoline, as an intermediate. The compounds of Formula VIare prepared from the intermediate, as indicated below.

The appropriate 4-bromo quinolines, 4-alkoxy quinolines and 4-thioalkoxyquinolines may be used in place of the 4-chloro quinolines to preparethe compounds of Formula VI. When these compounds are used, the reactionduration and conditions may slightly differ from those used when the4-chloro quinolines are the starting compounds. Those skilled in the artWill easily be able to determine the appropriate reaction conditions foruse where the 4-bromo, 4-alkoxy or 4-thioalkoxy quinolines are used asreactants. The preferred reactants are the 4- chloro quinolines.

Compounds VII, VIII and IX may be prepared from the appropriate 1chloro-6,7-substituted isoquinolines. These latter compounds may beprepared by a method derived from that given by E. L. Anderson et al.,J. Am. Pharm. Assoc., Sci. Ed., 41, 643 (1952).

The preparation method involves the reaction of a substitutedphenethylamine with ethyl chloroformate, in benzene, to form thesubstituted phenethyl carbamic acid, ethyl ester. This latter compoundis cyclized by treatment with polyphosphoric acid at about 140 C. for 30minutes to form the 3,4-dihydro-1(2H)-isoquinolinone which is convertedto the 1(2H) isoquinolinone by use of a palladium on carbon catalyst.This reaction is continued until hydrogen evolution ceases. The1(2H)-isoquinolinone is converted to the 1-chloro-isoquinoline byreaction with phosphorus oxychloride. The l-chloro-isoquinoline isreacted with the appropriate amine to form the compounds of FormulaeVII, VIII, and IX.

A typical reaction scheme may be shown as:

1 A- -CH2&HNH2 O t --o B benzene I A OHZHNHCO2Et polyphosphorlc acid---a B 140 C 30 minute A q-Ri Pd/C A -R1 P NH 240 0. NH B B reflux Y Y 1hour As is apparent from the reaction scheme set out above, thesubstituents at the 3-, 6- and 7-positions (R A and B) are determined bythe nature of the original substituted phenethylamine and the aminosubstituent at the l-position is determined by the amine used in thefinal reaction step. In the compounds of Formula IX, where thesubstituents R R R R and J are complex, it may be necessary orpreferable to form the final compound in two or more steps, from thel-chloro compound, as is outlined below.

The amination of the l-chloro isoquinoline is generally carried out inan aqueous or organic solvent and, while ethanol is preferred as asolvent, other polar solvents such as dimethylformamide, dioxane,tetrahydrofuran or methanol may be used. A molar excess of amine or basemay be used advantageously. The amination reaction takes place at atemperature ranging from 100 C. to 200 C. and a reaction duration of to36 hours. Preferred reaction temperatures are 120 C. to 140 C. andpreferred reaction times range from 16 to 24 hours when ethanol is usedas a solvent.

The compounds of Formulae VII, VIII and IX may also be produced usingthe appropriate l-bromo isoquinoline, the l-alkoxy isoquinoline or thel-thioalkoxy isoquinoline in place of the preferred l-chloro compound.Where these compounds are used in place of the l-chloro isoquinolines,the reaction conditions may differ from those given above for thel-chloro compound. Those skilled in the art will easily be able todetermine appropriate reaction temperatures and durations for producingthe compounds of Formulae VII, VIII and IX from the 1bromo, l-alkoxy orI-thioalkoxy isoquinolines.

The methods used to prepare the compounds of Formulae VII, VIII and IXmay also be used to prepare compounds of the following Formula X:

where A, B and R are as defined for their compounds of Formulae VII,VIII and IX and R R and R are each H or alkyl having up to 6 carbonatoms. The number of methylene groups, In, is from 2 to 5. Thesecompounds are useful as intermediates in preparing the compounds ofFormulae VII, VIII and IX.

Where compounds of Formulae II, IV, V, VA, VI and IX are produced it isoften preferable to prepare the final compound from the chloroderivative in two or more steps.

Nevertheless, the final compound may, in some cases, be more easilyprepared in a single step by direct amination of the chloro derivative.Thus, the 4-(2-alkyl-6,7-dimethoXyquinazolin-4-yl)-piperazine 1carboXylic acid, isobutyl esters are prepared, directly, by reacting the2- a1kyl-4-chloro-6,7-dimethoxyquinazolines with piperazinel-carboxylicacid, isobutyl ester. Similarly, the 4-(6,7-dimethoxyquinolin-4-yl)-piperazine-1-carboxylic acid, isobutyl ester isprepared, directly, by reacting 4-chloro-6,7- dimethoxyquinoline withthe appropriate ester.

Suitable amino compounds, useful for producing the compounds of thisinvention, in one or more steps, from the chloro, derivatives may beprepared by the following scheme:

In these reactions X is chloro or bromo. The last compound is reacteddirectly with the 4-chloro quinazoline, the 4-chloro-quinoline or thel-chloro-isoquinoline to form the compounds of this invention where I ischloro or bromo.

The compound where I is chloro or bromo may then be converted to thecorresponding compound, where J is hydroxyl, by treatment with 0.1 Nhydrochloric acid.

The compound where J is hydroxyl may then be converted to the compoundswhere J is acyloxy or aroyloXy by use of the appropriate acid chloride.

The compounds of Formulae II, IV, V, VA, VI, and IX may also be preparedin several stages, analogously to the scheme shown below:

Compounds where I alkylamido, arylamido or formamido may be producedfrom compounds where J is unsubstituted amino by treatment of the lattercompounds with the appropriate acid chloride.

Variations of these procedures which also may be used to prepare thecompounds of this invention from other similar compounds and by othermethods will be obvious to those skilled in the art.

The well-known procedures for preparing salts of basic compounds arealso applicable to the preparation of the compounds of this inventionand are illustrated in the examples below. Such salts may be formed withboth pharmaceutically-acceptable and pharmaceutically-unacceptableacids. By pharmaceutically-acceptable is meant those salt-forming acidswhich do not substantially increase the toxicity of the basic compound.The preferred salts, which are of particular value in therapy, are theacid addition salts. These include salts of mineral acids such ashydrochloric, hydriodic, hydrobromic, phosphoric, metaphosphoric, andsulfuric acids, as well as salts of organic acids such as tartaric,acetic, citric, malic, maleic, methanesulfonic, ethansulfonic,benzenesulfonic, paratoluenesulfonic and gluconic as well as othersuitable acids.

The pharmaceutically-unacceptable acid addition salts, while not usefulfor therapy, are vulable for use in the isolation and purification ofthese newly discovered compounds. Furthermore, they are useful for thepreparation of the therapeutically valuable pharmaceutically-acceptablesalts. Of this group, the more common salts include those formed withhydrofluoric and perchloric acids. Hydrofluoride salts are particularlyuseful for the prepara tion of the pharmaceutically-acceptable salts.

The compounds of this invention may be administered to hypertensivesubjects or subjects suffering from bronchoconstriction in order toalleviate these conditions. The bronchoconstriction may be functional ormay be caused by allergenic conditions or asthmatic conditions or mayhave come about as the result of a microbial infection. The compounds ofthis invention may be administered alone or in combinations withpharmaceutically-acceptable carriers. The proportion of the activeingredient to carrier is determined by the solubility and chemicalnature of the therapeutic compounds, the

chosen route of administration and the needs of the standardpharmaceutical practice. For example, where these compounds areadministered in tablet form, excipients such as lactose, sodium citrate,calcium carbonate and dicalcium phosphate may be used. Variousdisintegrants such as starch, alginic acids, and certain complexsilicates, together with lubricating agents such as magnesium stearate,sodium lauryl sulphate and talc, may also be used in producing tabletsfor the oral administration of these compounds. For oral administrationin capsule form, lactose and high molecular weight polyethylene glycolsare preferred materials for use as pharmaceutically-acceptable carriers.Where aqueous suspensions are to be used for oral administration, thecompounds of this invention may be combined with emulsifying orsuspending agents. Diluents such as ethanol, propylene glycol, glycerineand their combinations may be employed as well as other materials.Solutions of the compounds of this invention in combination with othersolutes such as glucose or saline may be used where the compounds are tobe administered parenterally. Such aqueous solutions should be suitablybuffered, if necessary, to render them isotonic.

The compounds of this invention may be administered to subjectssuffering from bronchoconstriction by means of inhalators or otherdevices which permit the active compounds to come into direct contactwith the constricted areas of the tissues of the subject.

The dosage required to reduce the blood pressure of hypertensivesubjects and to relieve the bronchoconstriction in affected subjectswill be determined by the nature and the extent of the hypertension orbronchconstriction. Generally, small dosages will be administeredinitially with gradual increase in dosage until the optimal dosage levelis determined for the particular subject under treatment. It willgenerally be found that when the composition is administered orally,larger quantities of the active ingredient will be required in order toproduce the same level of blood pressure reduction orbronchoconstriction relief as would be produced by the smaller quantityof active compound which is administered parenterally. In general,dosages will be in the range from about 0.02 to 200 milligrams of activeingredient per kilogram of body weight of subject, administered insingle or multiple dosage units. Dosages at this level will effectivelyreduce blood pressure in hypertensive subjects and relievebronchoconstriction in subjects suffering therefrom. Tablets containing0.1 to 50 milligrams of active ingredient are found to be particularlyuseful.

The therapeutic effects of the compounds of this invention have beenevaluated in guinea pigs and dogs. It was found that the compoundsmanifested therapeutic action over extended periods of time, were easilyabsorbed into the treated subjects upon administration and showedrelatively small effects on the central nervous systems of the treatedsubjects. The effectiveness of the compounds of this invention astherapeutic agents was measured with respect to theophylline, a knownbronchodilator and with respect to known hypotensive agents.

The compounds of this invention also have been observed to inhibit theactivity of the enzyme phosphodiesterase, which catalyzes the conversionof adenosine-3', 5,-monophosphate (3',5'AMP) toadenosine-5-monophosphate (5-AMP). Thus, in systems containingphosphodiesterase in which it is desirable to maintain a high 3',5AMPlevel, the instant compound might be used to great advantage. Theability of the novel compounds to inhibit the enzyme activity is ofsignificance since it is well known that the mononucleotide 3',5AMP isan important regulator of numerous cellular and tissue processes, e.g.smooth muscle relaxation, lipolysis and glycolysis. The instantcompounds are tissue-specific inhibitors of the enzyme, i.e., they willinhibit the enzyme in certain tissues and not in others; therefore, whenit is desirable to raise the 3',5'AMP level in only one of several typesof tissues which are present, use of the instant compounds isparticularly advantageous.

This phosphodiesterase inhibition is also significant for the relaxationof bronchial and peripheral vascular smooth muscle. Many of the novelcompounds have been evaluated with respect to their ability to inhibitphosphodiesterase activity with a view to their potential activity asbronchodil-ators, smooth muscle relaxants or anti-hypertensives.

It will be understood that various changes in the details, materials andsteps which have been herein described and illustrated in order toexplain the nature of this invention may be made by those skilled in theart within the principle scope of the invention.

The following examples are given by way of illustration only and are notto be construed as limiting the scope of this invention in any way.

EXAMPLE I Preparation of 4-(Z-ethyl-6,7-dimethoxyquinazoline-4-yl)-piperazine-l-carboxylic acid, isobutyl ester PART A.--PREPARATION OFN-CARBETHOXY-G- AMINOVERATRIC ACID A quantity of ethyl chloroformatetotaling 20.0 grams (0.185 mole) was added, dropwise, to a solution of35.0 grams (0.177 mole) of 6-aminoveratric acid dissolved in 100 ml. ofpyridine. The addition was ac complished over a 30 minute period and thetemperature of the reacting mixture was always held below C. Theprecipitation of the product began after about half of the dropwiseaddition had taken place. After completion of the addition, the mixturewas cooled to room temperature and poured into 400 ml. of water. Thismixture was filtered, washed with three 200 ml. portions of water, anddried over phosphorus pentoxide. The procedure yielded 46.0 grams ofN-carbethoxy-6-aminoveratric acid as a white solid with a melting pointof 211213 C. The procedure produced a yield of 96.5 percent.

PART B.PREPARATION OF 6-AMINOVERATRAMIDE A slurry of 46.0 grams (0.171mole) of N-carbethoxy- 6-aminoveratric acid in ml. of thionyl chloridewas stirred until the exothermic reaction had subsided. The mixture wasthen heated for 15 minutes at reflux. The slurry was diluted with hexaneand refluxed for 15 minutes after which the resultant mixture wasfiltered to yield 45.0 grams of crude 4,5-dimethoxy-N-carboxyanthranilicanhydride. The crude product of this step evidenced a melting point of225-256 C. and was used, without purification, as follows.

A slurry of 35.0 grams of the crude 4,5-dimethoxy-N- carboxyanthranilicanhydride with 600 ml. of ethanol saturated with ammonia was stirred atreflux for 30 minutes while a stream of ammonia was passed through themixture. A complete solution resulted after 15 minutes. The solution wascooled to room temperature and filtered and the filtrate wasconcentrated to separate 25.4 grams of crystalline fi-aminoveratramide.This sample evidenced a melting point of 133-136" C. Furthercrystallization of the product from benzene yielded an analytical samplewhich evidenced a melting point of 147148 C.

Analysis.Calcd for C H H O (percent): C, 55.09; H, 6.17; N, 14.28. Found(percent): C, 55.24; H, 5.83; N, 14.54.

PART C.PREPARATION OF (S-PROPIONAMIDO- VERATRAMIDE A dropwise additionof 10.1 grams (0.1 mole) of propionyl chloride was made to a solution of19.6 grams (0.1 mole) of 6-amino-veratramide in 50 ml. of pyridine. Thesolution was held at a temperature below 10 C. during the addition.Precipitation was initiated about 5 minutes after the completion of theaddition and the 9 resultant mixture was permitted to attain roomtemperature. The mixture was combined with 400 m1. of water, withconstant stirring, and the crystalline product was removed byfiltration. The filter cake was washed with water and then dried overphosphorus pentoxide. This procedure afforded 21.6 grams of6-propionamido-veratramide which was separated as a white amorphoussolid. The melting point of this solid was 196-197 C. and it representeda yield of 86.5 percent. Recrystallization of a 2.5 gram portion of thewhite solid from 60 ml. of methanol yielded 2.3 grams of white crystalswhich were used as the analytical sample. The recrystallized sample hada melting point of 196-197 C. and was found to be homogeneous by thinlayer chromatography.

Analysis.-Calcd for C H N O ,(percent): C, 57.13; H, 6.39; N, 11.11.Found (percent): C, 57.09; H, 6.27; N, 11.04.

PART D.PREPARATION OF 2ETHYL-6,7-DIMETHOXY- 41(3H) -QUINAZOLINONE\' Amixture containing 17.1 grams (0.068 mole) of 6-propionamido-veratramide, 4.45 grams (0.079 mole) of potassium hydroxideand 100 ml. of absolute ethanol was stirred and refluxed under anitrogen atmosphere for 90 minutes. The resultant clear yellow solutionwas evaporated to remove the bulk of the ethanol and the residue wasadded to 500 ml. of water and acidified with glacial acetic acid to pHof 6. The mixture was filtered and the filter cake was washed withethanol and water. The solid was dried over phosphorus pentoxide and16.0 grams of product were obtained as a white, microcrystalline solid.The white solid, which represented a yield of 100 percent, had a meltingpoint of 252-253 C. and was homogeneous by thin layer chromatography.

PART E.PREPARATION F 2-ETHYL-4-CHLORO-6,7-

DIMETHOXYQUINAZOLINE A mixture of 16.0 grams (0.068 mole) of2-ethyl-6,7- dimethoxy-4-(3H)-quinazolinone in 160 ml. of phosphorusoxychloride was stirred with reflux for 3.5 hours. The resultingsolution, which was formed after 2.5 hours of stirring at reflux, wasconcentrated to an oily residue which was dissolved in 500 ml. ofmethylene chloride. This solution was added to 500 ml. of concentratedammonium hydroxide solution with constant stirring. The methylenechloride layer was drawn off and the resultant aqueous phase wasextracted with two 200 ml. portions of methylene chloride. The combinedorganic phases were washed with two 200 ml. portions of water and driedover sodium sulfate. Evaporation of the methylene chloride yielded 15.3grams of 2 ethyl-4-ch1oro-6,7-dimethoxyquinazoline as a homogeneousyellow amorphous solid. The solid evidenced a melting point of 150-154C. and represented a yield of 89 percent. An analytical sample wasobtained by recrystallization from a methylene chloride-hexane mixture.The product, which was separated as small colorless needles, evidenced amelting point of 155- 156.5 C. and was homogeneous by thin layerchromatography.

PART I l-PREPARATION OF 4 (2 ETHYL-G,7-DIME- THOXYQUINAZOLIN 4 YL)-PIPERAZINE-1-CAERBOX- YLIC ACID, ISOBUTYL ESTER A mixture of 5.0 grams(0.02 mole) of 2-ethyl-4-chloro- 6,7-dimethoxyquinazoline and 7.43 grams(0.04 mole) of piperazine-l-carboxylic acid, isobutyl ester wasdissolved in 50 ml. of absolute ethanol and was stirred at reflux for 1hour. The resultant clear solution was concentrated to a crystallineresidue and mixed with 150 ml. of boiling hexane. The resultant mixturewas filtered and the filtrate was concentrated. On cooling, 6.4 grams of4-(2-ethyl-6.7- dimethoxyquinazolin 4-yl)-piperazine-1-carboxylic acid,isobutyl ester was deposited as a white microcrystalline solid whichevidenced a melting point of 98-l01 C. and was homogeneous by thin layerchromatography. This amount represented a yield of 79.5 percent. Ananalytical 10 sample was obtained by a further recrystallization fromhexane to yield a product comprised of white needles which evidenced amelting point of 96-97 C.

EXAMPLE II Preparation of 4-dimethylamino-6,7-dimethoxyquinazoline PARTA.PREPARATION 0F 6,1-DIMETHOXY-4-(3H)- QUINAZOLINONE The procedures ofPart A and Part B of Example I were carried out to prepare6-aminoveratramide.

A mixture of 25.4 grams (0.129 mole) of 6-aminoveratramide and 50 ml. of97 percent formic acid was stirred at reflux for 2 hours to yieldcomplete dissolution of the solid. Upon cooling to room temperature,crystallization occurred. The mixture was diluted with 200 ml. ofethanol and filtered. The filter cake was dried and 21.5 grams of whitesolid was obtained. The white solid, which evidenced a melting point of293-294 C., was homogeneous by thin layer chromatography. The yieldobtained by this procedure was 81 percent. An analytical sample wasobtained by recrystallization from dimethylformamide and white needles,which melted at 296-298 C., were obtained.

Analysis.-Calcd. for C H N 0 (percent): C, 58.25; H, 4.89; N, 13.58.Found ,(percent): C, 58.10; H, 5.01; N, 13.49.

The hydrochloride was prepared by the method of Example H and evidenceda melting point of 260-261 C.

Analysis.--Calcd. for clgHmNgogHcl (percent): C, 49.49; H, 4.57; N,11.54; Cl, 14.61. Found (percent): C, 49.68; H, 4.76; N, 11.33; C1,14.85.

PART B.-PREPARATION OF 4-CHLOR0-6,7-DI- METHOXLQUINAZOLINE' A mixture of21.5 grams (0.104 mole) of 6,7-dimethoxy-4(3H)-quinazolinone and 215 ml.of phosphorus oxychloride was stirred at reflux for 4 hours. A completesolution resulted after one hour. The solution was concentrated to anoily residue which was added slowly to 400 ml. of concentrated ammoniumhydroxide solution. The resultant solid was filtered, washed well withWater and dried in air to yield 19.5 grams of solid material whichevidenced a melting point of 181-185 C. Upon recrystallization fromethanol, 14.5 grams of a white crystalline product, which evidenced amelting point of 184-186 C was produced in a yield of 62.5 percent.

PART C.PREPARATION OF 4-D'IMETHYLAMINO-6fl- DIMETHOXYQUINAZOLINE Astainless steel pressure bottle was charged with 4.0 grams (0.0179 mole)of 4-ch1oro-6,7-dimethoxyquinazoline and ml. of a 1:2dimethylamine-ethanol solution. The bottle was heated at C. for 4 hours.Upon cooling, 1.7 grams of white crystalline 4-dimethylamino-6,7-dimethoxyquinazoline was deposited. The product, which manifested amelting point of 157-159" C., was obtained in a yield of 41 percent. Thefiltrate was evaporated to dryness and the crude residue, which totalled3.3 grams, was eluted from a Florisil column with benzene (7 x ml.fractions) to separate a further yield of 1.2 grams of crystallinematerial which mainifested a melting point of 158- 160 C. This wasidentical with the first product crop and both crops showed a singlespot upon thin layer chromatography. The total yield for this procedurewas 70 percent.

Analysin-Calcd. for C H N O ,(percent): C, 61.78; H, 6.48; N, 18.02.Found (percent): C, 61.97; H, 6.37; N, 18.05.

EXAMPLE III Preparation of 4-piperazinyl-6,7-dimethoxyquinazoline Theprocedure of Parts A and B of Example II were used to prepare4-chloro-6,7-dimethoxyquinazoline.

A solution of 20.5 grams (0.09 mole) of 4-chloro-6,7-dimethoxyquinazoline dissolved in 1 liter of chloroform 1 l was added,dropwise, over a 24 hour period, to a stirred, refluxing solution of77.4 grams (0.9 mole) of anhydrous piperazine dissolved in 1 liter ofabsolute ethanol. The resultant solution was evaporated to a crystallineresidue which was dissolved in 600 ml. of water. The aqueous solutionwas extracted with four 200 ml. portions of methylene chloride and thecombined extracts were dried over sodium sulfate. Evaporation of thesolvent alforded 23.7 grams of a crystalline residue which melted atl43-147 C. The residue was recrystallized from 150 ml. of ethyl acetateto separate 18.2 grams of white rods, with a melting point of 147-148"C., which were homogenous by thin layer chromatography. Furtherrecrystallization from ethyl acetate provided an analytical sample, aswhite rods, which melted at 150-1515 C. The procedure afforded a yieldof 74 percent.

Analysis.Calcd. for C H N O (percent): C, 61.29; H, 6.61; N, 20.43.Found (percent)3: C, 61.24; H, 6.4-0; N, 20.26.

EXAMPLE IV 2 Bronchodilator activity Conscious female guinea pigs, whichhad been fasted for 12 hours, recived oral or parenteral dosages of thecompound which was to be tested for effectiveness. Control animalsreceived doses of saline solution which did not contain the compoundwhich was under test. Subsequent to this administration, each animal waschallenged with histamine aerosol.

The challenge procedure consisted of spraying a 0.4 percent aqueoussolution of histamine, at a pressure of 5 lb./in. into an 8 x 8 x 12inch plastic container for one minute. Immediately after the containerwas subjected to the histamine spray the animal was placed within it. Atthe end of one minute of exposure, the

respiratory status, which is a reflection of bronchoconstriction, wasevaluated. Evaluation levels were designated and scored as normalbreathing (0), slightly deepened breathing 1), labored breathing (2),severely labored breathing and ataxia (3) and unconsciousness (4). Eachgroup of animals contained 8 to 10 individuals and a control groupcontaining the same approximate number was used. The scores for thecontrol group and the group which had been treated with the compoundunder test were compared and the diflerence was expressed as percentprotection.

The doses, which were given orally, were 60 mg./ kg. and the animalswere challenged with histamine 60 minutes later. The standard compoundused was theophylline which gave 25 percent protection after a dose of 60 mg./kg. was administered orally and the animal was challenged one hourlater. When the compounds listed in Table VI below were administeredaccording to this procedure and the animals were challenged accordingly,the

following percent protection was observed.

TABLE VI Percent protection C H 27 CH CH OOC- 28 CH CH CH OOC 40 CH (CHOOC 14 (CH CHCH OOC- 50 CH (CH OOC 20 CH (CH OOC 13 CH OC 16 CH (CH OC23 (Cl) COC 16 Percent protection cH,=cH 56 CH3OH2CH2- 53 (CH CH- 3 aAfter 1'5 minutes.

A B R R3 Percent protection Cyclopropyl- CH N /Rs CHaO- N N CHsO PercentB R: R: protection CH;-- 11-- H 41 113- CH3CHz- 0113011? 53 CH; H3- a-27 N onto N N-Rr OHIO N I OH:

Percent R protection 22 ClHsC- C'HsCHzOOO- 41 CHzCHCHa- 16 'EXAM'PLEVPreparation of 4-(6,7-dimethoxyquinazolin-4-yl)-piperazine-l-carboxylicacid, 2-methyl-2-ch1oropropyl ester To a cold C.) solution of 2.74 g.(0.010 mole) of 4-piperazinyl-6,7-dimethoxyquinazoline, prepared by themethod of Example III, and 1.10 g. (0.010 mole) of triethylamine in 40ml. of methylene chloride was added a solution of 1.7 g. (0.010 mole);of 2-rnethyl-2-chloropropyl chloroformate in 20 ml. of methylenechloride, dropwise, over 10 minutes. After warming to room temperature,the cloudy mixture was diluted with 50 ml. of methylene chloride, washedwith 50 m1. of water and dried over sodium sulfate. Evaporation of thesolvent left 4.3 g. of white crystalline solid, M.P. 140-146 C.Recrystallization from acetone-water mixture produced 2.6 g. (63.5%) ofthe analytical sample as a white crystalline solid, M.P. 158-159 C.

Analysis.-Calcd. for C H O N Cl (percent): C, 55.80; H, 6.19; N, 13.70;Cl, 8.67. Found (percent): C, 55.50; H, 6.15; N, 13.77; Cl, 8.59.

EXAMPLE VI Preparation of4-(6,7-dimethoxyquiuazolin-4-yl)-piperazine-l-carboxylic acid,2-methyl-2-hydroxypropyl ester A solution of 6.0 g. (0.0147 mole) ofcrude 4-(6,7- dimethoxyquinazolin-4-yl) -piperazine-l-carboxylic acid,2- methyl-2-chloropropyl ester in 225 ml. of 0.1 N hydrochloric acid wasallowed to reflux for 1 hour. The cooled solution was extracted withthree 100 m1. portions of methylene chloride, and the aqueous phasesubsequently made basic with dilute sodium hydroxide solution andextracted with three 100 ml. portions of methylene chloride. Theselatter organic extracts were combined, dried over sodium sulfate andevaporated and the resulting residue recrystallized from achloroform-ethyl acetate mixture to separate 2.1 g. (37%) of whiteprisms M.P. 199- 220 C.

Analysis.-Calcd. for C H O N (percent): C, 58.45; H, 6.71; N, 14.35.Found (percent): C, 58.36; H, 6.63; N, 14.62.

14 EXAMPLE v11 Preparation of4-(6,7-dimethoxyquinazolinl-yl)-piperazine-l-carboxylic acid,2-methyl-2-propenyl ester, hydrochloride A mixture of 40.4 g. (0.18mole) of 4-chloro-6,7-dimethoxyquinazoline, prepared by the method ofPart B of Example II, and 33.0 g. (0.18 mole) of piperazine-lcarboxylicacid, 2-methyl-2-propenyl ester in 400 ml. of ethanol was refluxed for 4hours, and then evaporated to dryness to leave 73 g. (100%) ofcrystalline solid. This was dissolved in 600 ml. of water and extractedwith four 500 ml. portions of methylene chloride. The combined extractswere evaporated to a volume of 500 m1. and ethyl acetate added untilcrystallization began. Filtration separated 48.0 g. (65%) of thehydrochloride salt of the product as a pale yellow microcrystallinesolid M.P. 212- 213 C. (dec.).

A second crop (6.2 g., 8.4%) of product as a yellow microcrystallinesolid, M.P. 210-213 C. (dec.) was obtained by concentration of thefiltrate. This material was homogenous by thin layer chromatography.

EXAMPLE VIII Preparation of 4-(6,7-dimethoxyquinazolin 4yl)-piperazine-1-carboxylic acid, Z-methyl 2 hydroxypropyl ester To 200ml. of 50% sulfuric acid-water mixture was added 25 g. (0.0615 mole) of4-(6,7-dimethoxyquinazolin-4-yl)-piperazine-1-carboxylic acid, 2- methyl2 propenyl ester, hydrochloride. The resulting yellow solution wasstirred at room temperature for 1 hour, poured into 200 g. of ice andmade basic with 40% sodium hydroxide solution, keeping the temperaturebelow 40 C. The resulting solution was extracted with four 200 ml.portions of methylene chloride, and the combined organic extracts wereextracted with 0.5 N hydrochloric acid and finally with three 100 ml.portions of water. The combined aqueous extracts were made basic with40% sodium hydroxide solution and extracted with three 200 ml. portionsof methylene chloride. The latter methylene chloride extracts werecombined, dried over sodium sulfate and evaporated to leave 18.0 g. ofcrystalline solid M.P. l92-195 C. This was recrystallized fromchloroform-ethyl acetate mixture to separate 14.5 g. of analyticallypure product M.P. 198-1995 C.

EXAMPLE IX Five parts, by weight, of the4-(6,7-dimethoxyquinazolin-4-yl)-piperazine-l-carboxylic acid, Z-methyl2 hydroxypropyl ester is dissolved in pyridine at 0 C. and an excess ofpropionyl chloride is added, dropwise. At the completion of theaddition, the resulting mixture is refluxed for 1 hour, cooled, dilutedwith water and extracted three times with methylene chloride. Afterwashing, the combined methylene chloride layers with dilute acid, theyare dried and evaporated to dryness to give4-(6,7-dimethoxyquiuazolin-4-yl)-piperazine 1 carboxylic acid, Z-methyl-2 propionoxypropyl ester which is purified by recrystallization ifnecessary.

In a similar manner, the Z-methyl-2-butyryloxypropyl ester, the2-rnethyl-2-uaphthoyloxypropyl ester, the 2- rnethyl-2-benzoxypropylester and the 2-methy1-2-acetoxy propyl ester are prepared.

EXAMPLE X Preparation of 4-(6,7-dirnethoxyquinazolin 4yl)-piperazine-1-carboxylic acid, Z-dimethylaminoethyl ester PARTA.-PREPARATION OF 4-(6,7,-DIMETHOXYQUIN- AZOLIN 4 YL)-PIPERAZINE-1-THIOLCARBOXYLIC ACID, PHENYL ESTER To a cold (0) solution of 20.0 g. (0.073mole) of 4- piperazinyl 6,7 dimethoxyquinazoline, prepared by themethods of Example III, and 8.05 g. (0.084 mole) of triethylamine in 150ml. of methylene chloride was added, dropwise, 12.9 g. (0.075 mole) ofphenyl chlorothiolformate. The resulting mixture was allowed to stir atroom temperature for minutes and 100 ml. of methylene chloride wasadded. This solution was washed with two 50 ml. portions of water, driedover sodium sulfate and evaporated to separate 27.46 g. of crudeproduct, M.P. 146-l5 1 C. Recrystallization from 500 ml. of methanolseparated 15.84 g. (53%) of product as o pale yellow microcrystallinesolid, M.P. 170-173 C. An additional 5.59 g. (19%), M.P. 166170 C., ofproduct was obtained from further concentration of the mother liquor.

PART B.-PREPARATION OF 4-('6,7-DIMETHOXYQUIN- AZOLIN-4-YL)-PIPERAZINE-1-CARBOXYLIC ACID, 2-DI- METHYLAMINOETHYL ESTER To asuspension of 1.60 g. (0.044 mole) of sodium hydride (60% mineral oildispersion) in 50 m1. of tetrahydrofuran was added 3.92 g. (0.044 mole(of 2-dimethylaminoethanol and the solution refluxed until gas evolutionceased (45 minutes). The resulting solution Was cooled to roomtemperature and a solution of 4.1 g. (0.01 mole) of4-(6,7-dimethoxyquinazolin-4-yl)-piperazine 1 thiol carboxylic acid,phenyl ester in 15 ml. of tetrahydrofuran was added at once and thesolution stirred at room temperature for 45 minutes. After dilution with50 ml. of water, the solution was concentrated to remove thetetrahydrofuran, and extracted with four 50 ml. portions of methylenechloride. The combined extracts were dried over sodium sulfate andevaporated to separate 4.39 g. of crude product, M.P. 1l0ll4 C.Recrystallization from ethylacetatehexane mixture gave 2.52 g. (65%) ofproduct as an off-white microcrystalline solid M.P. 100- 104 C.

The corresponding hydrochloride evidenced a melting point of 230232 C.

In a similar manner, the compound4-(6,7-dimethoxyquinazolin-4-yl)-piperazine 1 carboxylic acid,Z-diethylaminoethyl ester was prepared, M.P. 9093 C.; recrystallizedfrom isopropyl ether; M.P. of hydrochloride 151 C.

EXAMPLE XI When the procedure of Example X are employed, using theappropriate aminoalkanol or aminocarbinol in the procedure of Part B ofExample X and the product of Part A of Example X, the4-(6,7-dimethoxyquinazoline- 4-yl(-1-carboxylic acid,Z-methyl-2-ethylmethylaminopropyl ester, the 2-methyl-2-aminopropylester, the Z-methyl- 2-anilinopropyl ester, the2-methyl-2-butylaminopropyl ester, the Z-methyl-2-m-tolylaminopropylester, the 2- methyl-2-naphthylarninopropyl ester, the2-methyl-2-pyrrolinylpropyl ester, the 2-rnethyl-2-piperidinylpropylester and the 2-methyl-2-hornopiperidinylpropyl ester are produced.

When the procedures of Example X are employed, using the appropriate2-alkoxycarbinol, 2-alkoxyalkanol, 2-aryloxycarbinol or 2-aryloxyalkanolin place of the 2- dimethylaminoethanol of Part B of Example X togetherwith the product of Part A of Example X, the4-(6,7-dimethoxyquinazolin-4-yl)-piperazine-1-carboxylic acid, 2-methyl-2-propoxypropyl ester, the 2-methyl-2-isobutoxypropyl ester, theZ-methyl-Z-phenoxypropyl ester, the 2- methyl-2-naphthoxypropyl esters,the Z-methyl-Z-xyloylpropyl ester and the Z-methyl-Z-methoxypropyl estermay be formed.

EXAMPLE XII Five parts, by weight of 4-(6,7-dimethoxyquinazolin-4-yl)-piperazine-l-carboxylic acid, 2-methyl-2-aminopropyl ester, preparedby the method of Example X, is dissolved in pyridine. To the solution isadded, dropwise, at 0 C., a slight excess of propionyl chloride. Afterthe addition is complete, the resultant mixture is heated at reflux. The

cooled mixture is diluted with water and the product 4-(6,7-dimethoxyquinazolin-4-yl)-piperazine 1 carboxylic acid,Z-methyI-Z-ethamidopropyl ester is removed by filtration or extractionof the aqueous solution with methylene chloride.

In a similar manner, the 2-methyl-2-formamidopropyl ester and theZ-methyl-2-butyramidopropyl ester are formed.

EXAMPLE XIII Preparation of4-(6,7-dimethoxyquinolin-4-yl)-piperazine-l-carboxylic acid, isobutylester A mixture of 3.4 g. (0.0152 mole) of4-chloro-6,7-dimethoxyquinoline, prepared by the general methods givenby B. Riegel et al., I. Am. Chem. Soc., 68, 1264 (1946), and 5.65 g.(0.031 mole) of piperazine-l-carboxylic acid, isobutyl ester in ml. ofethanol was heated at C. for 2 hours. The resulting solution wasconcentrated to dryness and the residue slurried in Water and filteredto separate 6.1 g. of crude product. Recrystallization from 50 ml. ofethanol afforded 4.3 g. (76%) of an off-white crystalline product, M.P.172173 C.

Analysis.-Calcd. for C H O N (percent): C, 64.32; H, 7.29; N, 11.25.Found (percent): C, 64.13; H, 7.16; N, 11.36.

EXAMPLE XIV Preparation of4-(6,7-dimethoxyquinolin-4-yl)-piperazinel-carboxylic acid,Z-methyl-Z-hydroxypropyl ester The methods used in Example VIII wereused to prepare the 2-methyl-2-hydroxypropyl ester which evidenced amelting point of 172-173 C. and was recrystallized from ethyl acetate.

EXAMPLE XV The methods of Examples V to XIV inclusive are used toprepare compounds of the following formulae:

N A Wm B The methods of Examples V to XIV inclusive are used EXAMPLE XVIto prepare compounds of the following formula:

I. 9 TABLE-Continued H H H H CHi H H H H /GH:

EXAMPLE XVII Preparation of 4-(6,7-dimethoxyisoquinolin-1-yl)-piperazine-l-carboxylic acid, isobutyl ester PART A.PREPARATION OFN-CARBETHOXYHOMO- VERATRYLAMINE To a solution of 362 g. (2.0 mole) ofhomoveratrylamine in 500 ml. dry benzene was added a solution of 109 g.(1.0 mole) of ethyl chloroformate in 200 ml. of henzene, dropwise,keeping the temperature at 50-55 C. The resulting slurry was stirred for16 hours at room temperature and poured into 500 ml. of water. Thebenzene layer was separated, washed with 500 ml. of saturated sodiumbicarbonate solution and dried over sodium sulfate. Evaporation ofsolvent and distillation of the residual oil afforded 186 g. (74%) ofclear colorless liquid; B.P. 174- 177 C. (0.2 mm.), which crystallizedon standing to give the product as a white crystalline solid, MP. 61-62C.

PART B.-PREPARATION OF 6,7-DIMETHOXY-3,4-DI- HYDRO-1 (2H)-ISOQUINOLINONE To one liter of stirred polyphosphoric acid was added330 g. (1.3 moles) on N-carbethoxyhomoveratrylamine. The resultingmixture was stirred at 140 C. for 30 minutes and poured onto 2 liters ofice-water. The aqueous solution was made basic with ammonium hydroxideand extracted several times with chloroform. The combined extracts weredried over sodium sulfate and evaporated to dryness. The residue wasrecrystallized from methylene chloride-ethyl acetate to afford 120 g.(44.5%) of white plates, M.P. 159-161 C.

PART C.PREPARATION OF 6,7 -DIMETHOXY-l(2H)- ISOQUINOLINONE A mixture of46.0 g. (0.222 mole) of 6,7-dirnethoxy-3, 4-dihydro-1(2H)-isoquinolinoneand 7.0 g. of palladium on carbon was stirred at 240 C. for minutes(when hydrogen evolution had ceased) under a nitrogen atmosphere. Themixture was cooled to room temperature and leached several times withhot chloroform. The combined chloroform extracts were concentrated todryness to afiord 39.0 g. (86%) of white crystalline product, M.P.228-232 C.

PART D.-PREPARATION OF I-CHLORO Gfl-DI- METHOXYISOQUINOLINE A mixturecontaining 39 g. (0.191 mole) of 6,7-dimethoxy-l(2H)-isoquinolinone and200 ml. of phosphorous oxychloride was stirred at reflux for 1 hour. Theresulting amber solution was concentrated to dryness and the residuedissolved in methylene chloride and added slowly to 100 ml. ofconcentrated ammonium hydroxide solution. The methylene chloride layerwas separated and the aqueous layer extracted with three 100 m1.portions of methylene chloride. The combined organic extracts were driedover sodium sulfate and evaporated to atford 35.0 g. (82%) of Whitecrystalline product, MP. 135-137 C.

20 AnaIysis.-Calcd. for C H NO cl (percent): C, 59.07; H, 4.51; N, 6.26;Cl, 15.85. Found (percent): C, 59.33; H, 4.60; N, 6.30; Cl, 16.06.

PART E.PREPARATION 0F 4-(6,7-DIMETHOXYISO- QUINOLIN-l-YL)PIPERAZINE-l-CARBOXYLIC ACID, ISOBUTYL ESTER A mixture containing 4.0 g.(0.018 mole) of l-chloro- 6,7-dimethoxyisoquinoline and 6.7 g. (0.036mole) of piperazine-l-carboxylic acid, isobutyl ester in ml. of ethanolwas heated at 130 C. for 16 hours in a closed vessel. The resultingamber solution was concentrated, slurried in water and filtered to give4.3 g. of crude product. This was recrystallized from 30 ml. of methanolto afford 2.58 g. (38.5%) of product as slightly pink needles, MP.130-132 C.

Analysis.-Calcd. for C20H27N304 (percent): C, 64.32; H, 7.29; N, 11.25.Found (percent): C, 64.47; H, 7.16; N, 11.29.

EXAMPLE XVIII Preparation of4-(6,7-dimethoxyisoquinolin-l-yl)-piperazine-l-carboxylic acid estersThe l-chloro-6,7-dimethoxyisoquinoline, produced by the methods of PartD of Example XVII was reacted with the appropriatepiperazine-l-carboxylic acid ester, according to the methods of Part Eof Example XVII to yield the following compounds:

4 (6,7 dimethoxyisoquinolin-lyl)-piperazine-l-carboxylic acid, ethylester; MP. 130131 C.; recrystallized from isopropyl ether; MP. ofhydrochloride salt 107- 108 C. (decomposition).

The methods of Example VIII were used to prepare the following compound:

4 (6,7 dimethoxyisoquinolin-1-yl)-piperazine-l-carboxylic acid,2-methyl-2-hydroxypropyl ester; MP. 133- 134 C.; recrystallized fromethyl acetate-hexane; MP. of hydrochloride salt C. (decomposition).

EXAMPLE XIX Preparation of 1-ethylamino-6,7-dimethoxyisoquinoline The1-chloro-6,7-dirnethoxyisoquinoline, produced by the methods of Part Dof Example XVII was reacted with ethylamine, according to the methods ofPart E of Example XVII, to yield 1-ethylamino-6,7-dimethoxyisoquiuoline,which evidenced a melting point of 194195 C. and was recrystallized frommethanol-water. The hydrochloride salt evidenced a melting point of224-225 C. (decomposition).

EXAMPLE XX Preparation of 1-piperazinyl-6,7-dimethoxyisoquinoline Asolution of 84.0 g. (0.243 mole) of 4-(6,7-dimethoxyisoquinolin-l-yl)-piperazine-1-carboxylic acid, ethyl ester, prepared bythe methods of Example XVIII, in one liter of methanol and 250 ml. of30% sodium hydroxide solution was refluxed for 18 hours. The resultingsuspension was concentrated to remove the methanol, diluted with 500 ml.of water and extracted with three 250 ml. portions of methylenechloride. The combined extracts were dried over sodium sulfate andconcentrated to 250 ml. To this was added 700 ml. of isopropyl ether andthe solution concentrated to 300 ml., chilled in an ice bath andfiltered to sepaarte 51 g. (77%) of white crystalline product M.P.134-135.5 C. An additional 15 g. (22.6%) of material was obtained byfurther concentration of the mother liquor.

EXAMPLE XXII Preparation of4-(6,7-dimethoxyisoquinolin-l-yl)-piperazine-l-carboxylic acid,Z-dimethylaminoethyl ester PART A.-PREPARATION OF 4-(6,7-DIMETHOXYISO-QUINOLIN-l-YL)-PIPERAZINE-1-THIOL CARBOXYLIC ACID, PHENYL ESTER To acold (0 C.) solution of 19.0 g. (0.0695 mole) of1-piperazinyl-6,7-dimethoxyisoquinoline, prepared by the methods ofExample XX, and 7.65 g. (0.076 mole) of triethylamine in 100 ml. ofmethylene chloride was added dropwise, 12.0 g. (0.0695 mole) of phenylchlorothiolformats. The resulting suspension was allowed to stir at roomtemperature for 15 minutes and diluted with 100 ml. of methylenechloride. This solution was washed with two 50 ml. portions of water,dried over sodium sulfate and concentrated to a crystalline residue. Theresidue was recrystallized from 100 ml. of methanol to separate 18.7 g.(66%) of product as a pale yellow crystalline solid, M.P. 137-138 C.

PART B.PREPARATION OF 4-(6,7-DIMETHOXYISO- QUINOLIN-l-YL)PIPERAZINE-l-CARBOXYLIC ACID, 2-DIMETHYLAMINOETHYL ESTER To a suspensionof 1.6 g. (0.044 mole) of sodium hydride (60% mineral oil dispersion) in100 ml. of tetrahydrofuran was added 3.92 g. (0.044 mole) of2-dimethylaminoethanol and the solution refluxed until gas evolutionceased (45 minutes). After cooling to room temperature 4.1 g. (0.01mole) of 4-(6,7-dimethoxyisoquinolin-l-yl)-piperazine-l-thiolcarboxylicacid, phenyl ester was added and the solution stirred at roomtemperature for 30 minutes. This was diluted with 70 ml. of Water andconcentrated to remove the tetrahydrofuran, and the resulting solutionextracted with three 100 ml. portions of methylene chloride. Thecombined extracts were dried over sodium sulfate and evaporated to leavea viscous oil which was triturated with isopropyl ether to separate 3.1g. (80%) of white crystalline product M.P. 114-115 C. Recrystallizationfrom methylene chloride-isopropyl ether mixture gave 2.3 g. of whitecrystalline product, M."P. 115 C.

EXAMPLE XXII The procedures of Examples V to XIV and XVII to XXIinclusive were used to produce the compounds of the following formulae:

otno B N CHSO I R R1 M.P. hydro- M.P. chloride, Recrystallization R Rbase, 0. C. solvent H CH(CH;): 138-140 200-204 Methanol. CH5 CH3 72-.75148-151 Purified by chromatography: CHhCH; CHgCH: 137-138.5 189-191Acetone-water.

CHaO- M1. hydro- M.P. chloride, Recrystallization G base,0. C. solvent--NCH; 163-166 220-225 Ethyl acetate. -N-CH 138-141 222-228 Methanol.

0 137-138 167-8 Methylene chloridelsepropyl ether. -NCCHa M 146-147 1135-7 Do. -N-C--CH:CHJ

l Decomposition.

The procedures of Examples V to VIII and XVII to XXII inclusive are usedto prepare compounds of the following formulae:

EXAMPLE XXIV Bronchodilator activity The procedures given in Example IVwere used to evaluate the bronchodilator activities of the followingcompounds. The percent protection was avaluated in the manner given inExample IV. The same dosage levels were used.

R H; R H; R CH CH Percent protection 14.

CHzO N\\ R,

N} N R1:

Percent 1 1: 15 J protection H H H H 20 H CH: CH: H 30 H C H; C H; O H62 This compound shows protection after 8 hours.

EXAMPLE XXV Spirally out strips of guinea pig trachea were prepared asdescribed by J W. Constantine, J. Pharm. PharmacoL, 16, 384 (1965).Isometric relaxations were recorded using a force displacementtransducer (Model FT-03, Grass Instrument Co., Quincy, Mass.) connectedto a Grass Model 7 polygraph (Grass Instrument Co., of Quincy, Mass).

The relative smooth muscle relaxing eflects of each of the compoundstested were compared as follows:

(1) The muscle relaxant effect of a bathing medium of 0.03 ,u.g./ml. ofisoproterenol was determined for each strip and was shown to besupramaximal. This response was taken as the maximum relaxation of thestrip.

(2) Subsequent relaxations of a given strip, to logarithmically-spacedconcentrations of the compounds under test, were determined and wereconverted to percent of isoproterenol-induced relaxation. Curves ofdosage vs. percent of maximum relaxation were obtained for eachcompound.

(3) The figure reported below for each compound was that concentration(expressed in ,agJrnil.) of compound which was needed to obtain 50% ofmaximum relaxation (EC of the tracheal strip. Consequently, the smallerthe E the more potent the compound was as a smooth muscle relaxant.

(4) Two standard compounds, theophylline (a known bronchodilator) andpapaverine, were evaluated as well.

The following compounds were tested with corresponding values of EC 1OECEO Theophylline 6.4 Papaverine 0.3

CHaO

CHaO

CHBO

30 EXAMPLE XXVI The compounds of this invention were evaluated withrespect to their power to inhibit the action of cyclic 3,5'-nucleosidephosphodiesterase which can act to destroy 3,5 adenosine monophosphate.

The cyclic 3,5-nucleoside phosphodiesterase was isolated using theprocedure of R. W. Butcher and E. W. Sutherland, J. Biol. Chem, 237,1244- (1962) and their purification procedure was carried through thethird step given, namely through the ammonium sulfate fractionation,dialysis and freezing steps, but not through the chomatographicfractionation step.

For each compound tested, two substrates, containing each of the twocontrol inhibitory compounds, and one substrate containing no inhibitorycompound were prepared. Each substrate had a total volume of 2 ml., was4 X molar in 3",5' adenosine monophosphate, contained 0.02 ml. of cyclic3,5-nucleoside phosphodiesterase and 4.0 rnoles 0t MgSO 0.2 ,amolesethylene diamine tetraacetate and 80 moles of a suitable buffer whichwas to maintain the pH at 7.5. Where the substrate also contained anovel compound whose phosphodiesterase inhibitory power was to be testedor contained a control inhibitory compound, the compound was present ata concentration of 10* molar.

Two control compounds, papaverine and theophylline, a knownbronchodilator, were run with each novel compound. Thus, at least foursubstrates, each containing 3,5 adenosine monophosphate, were run foreach novel compound evaluated. One contained the novel compound anothercontained theophylline, another contained papaverine and the lastcontained no phosphodiesterase inhibitor at all.

Each substrate was incubated for minutes at 30 C. after which thereaction was stopped by boiling for 10 minutes. At this point one mg. oflyopllilized Crotolus atrox venom dissolved in one ml. of pH 7.5 bufferwas added and the new mixture was incubated for 30 minutes at 30 C. andthis reaction was also stopped by boiling for 10 minutes. The venomreacts with 5-adenosine monophosphate, a product of the reaction betweenphophodiesterase and 3',5'-adenosine monophosphate, to release inorganicphosphate. Thus, a low final concentration of inorganic phosphateindicates that a small amount of 5'- adenosine monophosphate was formedand, therefore, that the phosphodiesterase activity was inhibited. Theinorganic phosphorus was determined colorimetrically by the methods ofC. H. Fiske and Y. Subbarow, J. Biol. Chem, 66, 375 (1925).

The percent inhibition was taken as the difference between the inorganicphosphate concentration in the substrate containing the inhibitingcompound and the concentration in the substrate containing no inhibitordivided by the concentration in the substrate without inhibitor.

The following compounds were tested:

Papaverine, percent inhibition N-iL-OCHzCH:

:1 0 & N CI-IaO I R Percent Theophyllino, Papaverine, inhibipercentpercent R tion inhibition inhibition 35 23 75 II --N N-C-OCHzCHa 0(I311; 55 23 75 -N IOCH2CH a (I? OH; 38 18 76 N N--C-OCHz-OE What isclaimed is:

11. A compound selected from the group consisting of 0 those of theformula who-$-14 14 I Ma and the pharmaceutically-acceptable mono-acidaddition salts thereof wherein A and B are each selected from the groupconsisting of methoxy, hydrogen, hydroxy and methyl with the provisothat when A or B is hydrogen, the other is never hydrogen; R R R R and Rare each selected from the group consisting of hydrogen and methyl and Jis selected from the group consisting of hydrogen, hydroxy, alkoxyhaving from 1 to 4 carbon atoms, chloro, bromo, and amino having up totwo sub- References Cited UNITED STATES PATENTS 2,593,798 4/1952Robinson 260-268 BQ 3,277,085 10/1966 Aebi et a1. 260-268 BQ 3,511,8364/1970 Hess 260-256.4 3,517,005 6/1970 Cronin 260268 BQ 2,637,699 1/1972Gabel et a1. 260256.4

DONALD G. DAUS, Primary Examiner US. Cl. X.R.

260268 BQ, 283 R, 283.8, 288 R, 284 R; 424-250, 258

